Compositions and methods for entrapping protein on a surface

ABSTRACT

The present invention provides a formulation to link protein to a solid support that comprises one or more proteins, Oligo-dT and one or more non-volatile, water-soluble protein solvents, solutes or combination thereof in an aqueous solution. Further provided is a method of attaching a protein to a surface of a substrate. The formulations provided herein are contacted onto the substrate surface, printed thereon and air dried. The substrate surface is irradiated with UV light to induce thymidine photochemical crosslinking via the thymidine moieties of the Oligo-dT.

CROSS-REFERENCE TO RELATED APPLICATION

This non-provisional application claims benefit of priority under 35U.S.C. §119(e) of provisional application U.S. Ser. No. 61/823,065,filed May 14, 2013, now abandoned, and to U.S. Ser. No. 14/120,278,filed May 14, 2014, the entirety of which are hereby incorporated byreference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates generally to the fields of microarrays andprotein chemistry. More specifically, the present invention relates to aformulation and methods for entrapping protein on a surface.

Description of the Related Art

It is well known that proteins may be attached to surfaces, typically bycovalent attachment of the protein directly to the solid substrate, orby covalent attachment to polymers that had previously been attached tothe surface, or by physical entrapment of the protein into pores withinthe solid surface itself, or by simple adsorption of the protein to thesurface of the microarray. Although such means of attachment allow for ahigher concentration of protein, there is a loss in functionality due tochemical modification of the surface. Sol-gels have been used to entrapproteins on solid supports. However, acceptable sol-gels are limited tothose without undesirable properties of gelling in the pin duringprinting, irreproducible spot sizes, cracking, poor adhesion,incompatibility with entrapped components, or reducing activity of theentrapped protein. None of these methods of attachment or entrappmentenable site-addressable, self-assembly of a 3 dimensional proteinstructure on a microarray.

Thus, there is a recognized need in the art for improved formulationsand methods for physically entrapping protein on a microarray surfacewithout direct attachment, binding or adsorption to the surface.Specifically, the prior art is deficient in aqueous crosslinkableformulations comprising Oligo-dT and protein(s) that can be entrappedand preserved in a native protein state in a high concentration on themicroarray. The present invention fulfills this longstanding need anddesire in the art.

SUMMARY OF THE INVENTION

The present invention is directed to a formulation to link protein to asolid support. The formulation comprises one or more proteins, Oligo-dTand one or more non-volatile, water-soluble protein solvents, solutes orcombination thereof in an aqueous solution.

The present invention also is directed to a formulation to link proteinto a solid support. The formulation comprises one or more proteins,Oligo-dT, and glycerol or glycerol and at least one of sucrose,trehalose or sorbitol in an aqueous solution.

The present invention is directed further to a method of attaching aprotein to a surface of a substrate. The method comprises contacting anaqueous formulation containing Oligo dT and a non-volatile, non-aqueoussolvent or solute or combination thereof and the protein onto thesubstrate surface and printing the formulation onto the surface. Thesubstrate surface is air dried and irradiated with UV light to inducethymidine photochemical crosslinking via the thymidine moieties of theOligo-dT, thereby attaching the protein to the surface of the substrate.

Other and further aspects, features, and advantages of the presentinvention will be apparent from the following description of thepresently preferred embodiments of the invention given for the purposeof disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages andobjects of the invention, as well as others that will become clear, areattained and can be understood in detail, more particular descriptionsof the invention briefly summarized above may be had by reference tocertain embodiments thereof that are illustrated in the appendeddrawings. These drawings form a part of the specification. It is to benoted, however, that the appended drawings illustrate preferredembodiments of the invention and therefore are not to be consideredlimiting in their scope.

FIG. 1A illustrates photocrosslinking to generate an Oligo-dT+Proteinmatrix on a microarray surface.

FIG. 1B illustrates the Oligo-dT+Protein matrix of FIG. 1A after UVcrosslinking.

FIG. 2A is a fluorescent microarray image of a bovine serum albumin(BSA) microarray on an amino silane surface.

FIG. 2B is a fluorescent microarray image of a BSA microarray on anepoxy silane surface.

FIG. 3 depicts SAPE (Cy-3) signals normalized to OligoT (Cy-5) signalsfrom a BSA microarray printed on an amino silane microarray surface, asin FIG. 2A.

FIG. 4 depicts SAPE (Cy-3) signals normalized to OligoT (Cy-5) signalsfrom a BSA microarray printed on an epoxy silane microarray surface, asin FIG. 2B.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term, “a” or “an” may mean one or more. As usedherein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one. Someembodiments of the invention may consist of or consist essentially ofone or more elements, method steps, and/or methods of the invention. Itis contemplated that any method, compound, composition, or devicedescribed herein can be implemented with respect to any other device,compound, composition, or method described herein.

As used herein, the term “or” in the claims refers to “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or”.

As used herein, the term “about” refers to a numeric value, including,for example, whole numbers, fractions, and percentages, whether or notexplicitly indicated. The term “about” generally refers to a range ofnumerical values, e.g., +/−5-10% of the recited value, that one ofordinary skill in the art would consider equivalent to the recitedvalue, e.g., having the same function or result. In some instances, theterm “about” may include numerical values that are rounded to thenearest significant figure.

In one embodiment of the present invention, there is provided aformulation to link protein to a solid support, comprising: one or moreproteins; Oligo-dT; and one or more non-volatile, water-soluble proteinsolvents, solutes or combination thereof in an aqueous solution. In oneaspect, the water soluble protein solvent may comprise glycerol or thewater soluble protein solvent and solutes may comprise glycerol and atleast one of sucrose, trehalose or sorbitol. In this aspect the sucrose,trehalose or sorbitol may be present at a mass ratio of about 0.5:1 upto about 4:1 relative to glycerol. In another aspect, the water solubleprotein solvent may comprise propanediol or the water soluble proteinsolvent and solids may comprise propanediol and at least one of sucrose,trehalose or sorbitol. In this aspect the mass ratio of the sucrose,trehalose or sorbitol is as described supra relative to propanediol. Inyet another aspect, the water soluble protein solvents and solids maycomprise glycerol and propanediol and at least one of sucrose, trehaloseor sorbitol. In yet another aspect, the formulation may be applied to ordisposed on a solid support such as an amino-silane layer upon anunderlying surface. In this aspect representative underlying surfacesmay be, but are not limited to a metal surface, a glass surface or aceramic surface. In yet another aspect, the formulation may be appliedto or disposed on a solid support such as an epoxy-silane layer upon anunderlying surface. In this aspect a representative underlying surfaceis a metal.

In this embodiment and aspects thereof, in the formulation of thepresent invention, the Oligo-dT may be about 30 bases to about 100 basesin length, preferably about 50 bases long and may be contained in theformulation in a concentration of at least 1 μg/ml, preferably about 1μg/ml (1 μM) to about 100 μg/ml (100 μM). Also, preferably, the proteinmay be about 50 kD to 250 kD in mass and may be contained in theformulation in a concentration of at least 10 μg/ml (10 μM). In additionthe non-volatile solvent and solutes may be formulated in water at about1% by mass. Furthermore, representative proteins which may be linked orattached to a surface include, but are not limited to an,immunoglobulin, a glycoprotein, a viral protein, an intact virus,albumin, an HLA, or an enzyme.

In another embodiment of the present invention, there is provided aformulation to link protein to a solid support, comprising one or moreproteins; Oligo-dT; and glycerol or glycerol and at least one ofsucrose, trehalose or sorbitol in an aqueous solution. In thisembodiment the glycerol, sucrose, trehalose and sorbitol may beformulated as described supra. In this embodiment and any aspectthereof, the Oligo-dT size and concentration, the protein size,concentration and type and the solid support all are as described supra.

In yet another embodiment of the present invention, there is provided amethod of attaching a protein to a surface of a substrate, comprisingthe steps of: contacting the aqueous formulation containing Oligo dT andone or more non-volatile, water-soluble protein solvents, solutes orcombination thereof and one or more proteins as described herein ontothe substrate surface; printing said formulation onto the substratesurface; air-drying the substrate surface; irradiating the substratesurface with UV light to induce thymidine photochemical crosslinking viathe thymidine moieties of the Oligo-dT, thereby attaching the protein tothe surface of the substrate. In aspects of this embodiment,representative water soluble protein solvents and solutes are as, andmay be formulated as, described supra. Also in this embodiment and allaspects thereof, the Oligo-dT size and concentration, the protein size,concentration and type and the solid support all are as described supra.

Described herein are methods and chemical formulations or compositionsto link proteins to a solid surface, such as a microarray surface, togenerate a biochemical or diagnostic binding assay. The surfaceattachment is produced by protein entrapment in a polymer network whichis formed around the protein, locally, by photo-crosslinking ofOligo-dT, a photoreactive polymer. Oligo-dT, plus one or more proteinsand one or more non-volatile, water-soluble solvents and solutes areapplied to the solid surface, locally, as a water solution. Uponapplication of that water solution to the solid surface, the watercomplement of the solution is allowed to evaporate away, yielding aconcentrated, water-depleted phase comprising Oligo-dT, protein,solvents, solutes which is then crosslinked, photochemically, to entrapthe protein within the resulting crosslinked, polymeric Oligo-dTnetwork. The non-volatile, water soluble solvents and solutes are chosenso that, subsequent to evaporative water loss, the resultingwater-depleted phase remains principally non-crystalline, therebymitigating protein damage by microcrystal formation. Proteins are linkedto an underlying surface, indirectly, rather than by direct chemicallinkage to the surface and in a way such that, subsequent to evaporativewater depletion and UV crosslinking, the protein becomes entrapped inthe crosslinked Oligo-dT network which was created around it. The abovecombination of indirect photochemical Oligo-dT network entrapment, plusretention of a non-crystalline phase upon water depletion, gives rise topreservation of a native protein state on the solid support, which isthen available, subsequent to rehydration, to bind to analytes appliedin water solution, as the basis for a binding, or diagnostic or publichealth screening assay.

More particularly, the Oligo-dT polymer chains are utilized as a linkermediating protein attachment to an underlying microarray surface.Oligo-dT is used a protein surface linker based on its capacity toengage in photocrosslinking. Briefly, Oligo-dT can readily be co-printedin @ 0.5× to 10× mole excess with any number of proteins of interest,e.g., albumin, antibodies, enzymes, HLA or any other water solubleprotein. At time of printing, the anionic Oligo-dT adsorbs,non-covalently, to the underlying cationic amino-silane surface viaformation of electrostatic bonds. If applied to an epoxysilane coatedsurface, it can associate with the surface via a combination of covalentlinkage to the epoxide and h-bonding to ring opened epoxide diols. Themicroarray spot is then allowed to air-dry over several minutes. In thepresent invention, protein printing occurs with one of severalwater-soluble (but non-volatile) solutes, in a buffered water solution,which upon air drying, becomes a water-depleted fluid, which retainssolubilization of the protein and eliminates buffer salt crystalformation, which would have occurred if the water-soluble non-aqueoussolutes were not added.

Several solutes and solute mixtures can be used, such as for example,including but not limited to glycerol; Glycerol with propanediol;propanediol; glycerol with sorbitol; glycerol with propane diol andsorbitol; propanediol and sorbitol; glycerol with trehalose; glycerolwith propane diol and trehalose; and propanediol and trehalose. Proteincan be printed at one of several concentrations (250 ug/ml-5 ug/ml); 500ug/ml; 250 ug/ml; 100 ug/ml; 50 ug/ml; 20 ug/ml; and 10 ug/ml.

In all cases, after printing and air-drying, the resulting microarrays(FIG. 1A) can be subjected to standard UV-Crosslinking at @300 mjoule,to photo-crosslink link the Oligo-dT (via T-T bonding) into aloosely-crosslinked matrix and in some cases to covalently link some ofthe protein to one or more nearby Oligo-dT molecules in thelocally-generated Oligo-dT matrix (FIG. 1B)

Subsequent to UV crosslinking, the microarray is then ready for use.Standard binding steps can be employed: to be performed at lab ambienttemperature or at elevated temperature: (1) Prebinding: Ordinarybuffered solution with a blocking agent to obscure unused surface sites;(2) Binding: Ordinary buffered solution with a blocking agent to obscureunused surface sites; (3) Washing: Ordinary buffered solution with ablocking agent to obscure unused surface sites; and/or (4) Dry andImage.

As would be immediately recognizable to a person having ordinary skillin this art, the formulations and methods of the present invention maybe used to fabricate a protein microarray via contact, or piezoelectricor ink jet printing onto a suitable solid support or to fabricate aprotein biosensor via contact. Alternatively, the formulation whenplaced in contact with an aqueous biological sample, the microarray maybe used as an in vitro diagnostic test or when placed in contact with anaqueous biological sample, the biosensor is used as an in vitrodiagnostic test.

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion.

Example 1 Structure of a Representative Microarray, to be Printed forthe Purposes of Testing Oligo-dT Entrapment of Protein on a MicroarraySurface

A single protein, for example, bovine albumin, can be printed to form amicroarray. Each spot in the microarray can differ in the proteinconcentration, the Oligo-dT concentration and the water soluble,non-volatile solute to be added (at @1% by mass) at time of printing.Buffer concentration can be as needed, but 2 mM Na₂Phos, pH 8.4 isrepresentative.

TABLE I Formulation of the individual microarray spots to vary Protein,Oligo-dT and Solvent composition in a 12 × 12 microarray Left 6 × 13segment of 12 × 12 Microarray Protein 250 250 100 100 50 50 conc ug/mlug/ml ug/ml ug/ml ug/ml ug/ml OligoT 50-mer 50-mer 50-mer 50-mer 50-mer50-mer 100 uM 100 uM 100 uM 100 uM 100 uM 100 uM Glycerol GlycerolGlycerol Glycerol Glycerol Glycerol OligoT 50-mer 50-mer 50-mer 50-mer50-mer 50-mer 50 uM 50 uM 50 uM 50 uM 50 uM 50 uM Glycerol GlycerolGlycerol Glycerol Glycerol Glycerol OligoT 50-mer 50-mer 50-mer 50-mer50-mer 50-mer 25 uM 25 uM 25 uM 25 uM 25 uM 25 uM Glycerol GlycerolGlycerol Glycerol Glycerol Glycerol OligoT 50-mer 50-mer 50-mer 50-mer50-mer 50-mer 100 uM 100 uM 100 uM 100 uM 100 uM 100 uM G-PD G-PD G-PDG-PD G-PD G-PD OligoT 50-mer 50-mer 50-mer 50-mer 50-mer 50-mer 50 uM 50uM 50 uM 50 uM 50 uM 50 uM G-PD G-PD G-PD G-PD G-PD G-PD OligoT 50-mer50-mer 50-mer 50-mer 50-mer 50-mer 25 uM 25 uM 25 uM 25 uM 25 uM 25 uMG-PD G-PD G-PD G-PD G-PD G-PD OligoT 50-mer 50-mer 50-mer 50-mer 50-mer50-mer 100 uM 100 uM 100 uM 100 uM 100 uM 100 uM PD PD PD PD PD PDOligoT 50-mer 50-mer 50-mer 50-mer 50-mer 50-mer 50 uM 50 uM 50 uM 50 uM50 uM 50 uM PD PD PD PD PD PD OligoT 50-mer 50-mer 50-mer 50-mer 50-mer50-mer 25 uM 25 uM 25 uM 25 uM 25 uM 25 uM PD PD PD PD PD PD OligoT50-mer 50-mer 50-mer 50-mer 50-mer 50-mer 100 uM 100 uM 100 uM 100 uM100 uM 100 uM Trehalose Trehalose Trehalose Trehalose TrehaloseTrehalose OligoT 50-mer 50-mer 50-mer 50-mer 50-mer 50-mer 50 uM 50 uM50 uM 50 uM 50 uM 50 uM Trehalose Trehalose Trehalose TrehaloseTrehalose Trehalose OligoT 50-mer 50-mer 50-mer 50-mer 50-mer 50-mer 25uM 25 uM 25 uM 25 uM 25 uM 25 uM Trehalose Trehalose Trehalose TrehaloseTrehalose Trehalose Right 6 × 13 segment of 12 × 12 Microarray Protein20 20 10 10 5 5 conc ug/ml ug/ml ug/ml ug/ml ug/ml ug/ml OligoT 50-mer50-mer 50-mer 50-mer 50-mer 50-mer 100 uM 100 uM 100 uM 100 uM 100 uM100 uM Glycerol Glycerol Glycerol Glycerol Glycerol Glycerol OligoT50-mer 50-mer 50-mer 50-mer 50-mer 50-mer 50 uM 50 uM 50 uM 50 uM 50 uM50 uM Glycerol Glycerol Glycerol Glycerol Glycerol Glycerol OligoT50-mer 50-mer 50-mer 50-mer 50-mer 50-mer 25 uM 25 uM 25 uM 25 uM 25 uM25 uM Glycerol Glycerol Glycerol Glycerol Glycerol Glycerol OligoT50-mer 50-mer 50-mer 50-mer 50-mer 50-mer 100 uM 100 uM 100 uM 100 uM100 uM 100 uM G-PD G-PD G-PD G-PD G-PD G-PD OligoT 50-mer 50-mer 50-mer50-mer 50-mer 50-mer 50 uM 50 uM 50 uM 50 uM 50 uM 50 uM G-PD G-PD G-PDG-PD G-PD G-PD OligoT 50-mer 50-mer 50-mer 50-mer 50-mer 50-mer 25 uM 25uM 25 uM 25 uM 25 uM 25 uM G-PD G-PD G-PD G-PD G-PD G-PD OligoT 50-mer50-mer 50-mer 50-mer 50-mer 50-mer 100 uM 100 uM 100 uM 100 uM 100 uM100 uM PD PD PD PD PD PD OligoT 50-mer 50-mer 50-mer 50-mer 50-mer50-mer 50 uM 50 uM 50 uM 50 uM 50 uM 50 uM PD PD PD PD PD PD OligoT50-mer 50-mer 50-mer 50-mer 50-mer 50-mer 25 uM 25 uM 25 uM 25 uM 25 uM25 uM PD PD PD PD PD PD OligoT 50-mer 50-mer 50-mer 50-mer 50-mer 50-mer100 uM 100 uM 100 uM 100 uM 100 uM 100 uM Trehalose Trehalose TrehaloseTrehalose Trehalose Trehalose OligoT 50-mer 50-mer 50-mer 50-mer 50-mer50-mer 50 uM 50 uM 50 uM 50 uM 50 uM 50 uM Trehalose Trehalose TrehaloseTrehalose Trehalose Trehalose OligoT 50-mer 50-mer 50-mer 50-mer 50-mer50-mer 25 uM 25 uM 25 uM 25 uM 25 uM 25 uM Trehalose Trehalose TrehaloseTrehalose Trehalose Trehalose OligoT 20 20 10 10 5 5 ug/ml ug/ml ug/mlug/ml ug/ml ug/ml Glycerol: Glycerol at 1% by mass; G-P:Glycerol-1,2Propanediol 1:1 at 1% by mass; PD: 1,2Propanediol at 1% bymass; Trehalose: Trehalose at 1% by mass

Example 2 Quantitative Considerations: Addition of Oligo-dT & ProteinPrior to Crosslinking on the Microarray Surface

It is well known that nucleic acid strands, especially those rich inthymidine, such as a simple repeating DNA oligomer dTn, i.e. Oligo-dT,become photochemically crosslinked to one another upon irradiation inthe 250 nm to 230 nm range, due to photochemical excitation of thethymidine ring, followed by addition to the C4-C5 bond of another T orC: or if in proximity, to a nearby protein, especially lysine orcysteine side chains or aromatic amino acid side chains tyrosine ortryptophan. The present invention exploits the photochemistry ofOligo-dT, or Oligo-U if an RNA oligomer, to create within a microarrayspot, local formation of a crosslinked Oligo-dT (Oligo-U) matrix whichcan physically entrap proteins which are applied along with Oligo-dT attime of microarray printing and before UV crosslinking (FIGS. 1A-1B).

Water soluble proteins in the 50 kD to 250 kD range are typically 10 nmto 20 nm in diameter. One can for example, employ bovine albumin, whichhas a mass of 67 kD and a measured nearly spherical radius of 7 nm. Therise per repeat of single stranded nucleic acids is @0.5 nm per base.Consequently a 50base long Oligo-dT molecule would present a totalcontour length of @25 nm, roughly 2× the diameter of the albumin.

If Oligo-dT were mixed in molar excess with a protein such as albumin,in a water solution that contained about 1% of a water-soluble fluidsolute, like glycerol, propanol, or glycerol-propanediol, or a watersoluble solid solute such as trehalose or sucrose or sorbitol, thenallowed to air-dry to evaporate away the water in it, the resultingwater-depleted phase will concentrate the protein and Oligo-dT moleculesin it about 100-fold. Below, several calculations are displayed for theresult of such concentration, for a representative 50 kd protein with adiameter of 14 nm which is similar to a globular protein such as bovinealbumin.

Microarray fabrication, of the kind to be exploited in the presentinvention is a type of nanotechnology, where individual microarrayelements or “spots” are applied to a surface as nanoliter droplets,which typically form spots on a surface that are about 100 microns indiameter. The present invention describes by calculation, the effect ofdepositing a typical 1 nanoliter droplet on a microarray surface, underconditions such that, as described above, air drying of the water in itwill cause non-volatile solutes, such as glycerol, or propanediol aloneor with solids like trehalose or sorbitol, to be concentrated 100-foldwithin the 100 um spot, thereby reducing the thickness of the spot.

Microarray Spot Fluid Thickness Calculation: Subsequent to Air-Drying

Assume 1×10⁻⁹ L print volume=1×10⁻⁸ cm³.

Assume 100-fold air-drying to 1×10⁻¹¹ L=1×10⁻⁸ cm³.

Assume Spot Diameter=1×10⁻² cm (100 um).

Spot Area=1×10⁻⁴ cm²

Film Thickness=Print Volume after Air-Drying/Spot Area=[1×10⁻⁸cm³]/1×10⁻⁴ cm²=1×10⁻⁴ cm=1 um

Assuming 1 nL print of 1% non-volatile solutes:

Final Spot Shape upon 100-fold evaporative concentration.

-   -   Spot Width=100 um and Spot Thickness=1 um

Surface Coverage Calculations

1). Assuming that a Protein is @14 nm in diameter, its 2D projection=150nm².

2). If the Microarray spot is 100 um wide, its area=0.75×10⁻⁸ m²=75×10⁺⁸nm².

3). So one 100 um spot will be covered by @0.5×10⁺⁸ Protein molecules,as a one-molecule thick layer

4). If contact print volume=1 nL, at:

-   -   5 uM=3×10⁺⁹ molecules per spot yields 30 molecule deep Protein        layer, post evaporation;    -   2 uM=1.5×10⁺⁹ molecules per spot yields 12 molecule deep Protein        layer, post evaporation;    -   1 uM=0.75×10⁺⁹ molecules per spot yields 1.5 molecule deep        Protein layer, post evaporation;    -   0.4 uM=0.3×10⁺⁹ molecules per spot yields 0.6 molecule deep        Protein layer, post evaporation;    -   0.2 uM=0.15×10⁺⁹ molecules per spot yields 0.3 molecule deep        Protein layer, post evaporation; and    -   0.1 uM=0.08×10⁺⁹ molecules per spot yields 0.15 molecule deep        Protein layer, post evaporation.

TABLE IIa Protein and Oligo-dT concentration subsequent to air-drying at1% of non-volatile solute to affect a controlled 100-fold volumedecrease Average Protein- Concentration Protein Molecular Protein PrintProtein Print After Drying Separation Upon Concentration Concentration100-Fold Drying 250 μg/ml   5 μM 500 μM  75 nm 100 μg/ml   2 μM 200 μM100 nm 50 μg/ml  1 μM 100 μM 130 nm 20 μg/ml 0.4 μM  40 μM 170 nm 10μg/ml 0.2 μM  20 μM 220 nm  5 μg/ml 0.1 μM  10 μM 270 nm

TABLE IIb Oligo-dT concentration subsequent to air-drying at 1% ofnon-volatile solute (100-fold volume decrease) Concentration AverageMolecular Oligo-dT Print After Drying Separation Upon Concentration100-Fold Drying 100 μM   10 mM 28 nm 50 μM  5 mM 35 nm 25 μM 2.5 mM 44nm 10 μM 1.0 mM 60 nm  2 μM 0.2 mM 100 nm   1 μM 0.1 mM 130 nm 

The calculations in Table IIa and Table IIb suggest that in an ordinarymicroarray spot that is @100 um in diameter, containing 1% by volume ofa non-volatile, water miscible solvent (like glycerol or propanediol ornon-volatile water-soluble solutes such as trehalose or sorbitol)ordinary air-drying will result in a spot that is about 1 um thick. Thecalculations also show that if a @50 kD protein is printed at @250ug/ml, the average separation between 14 nm wide protein molecules willbe reduced by concentration to @75 nm, or about 5× the protein diameter(FIG. 1A). If printed at @10 uM, Oligo-dT will also concentrate 100fold, to yield an average separation of only about 60 nm, or about 4×the diameter of the protein, thus yielding a dense network of Oligo-dTmolecules surrounding the protein molecules between them (FIG. 1A). Ifdesired, the effective pore size of the Oligo-dT network could beincreased to an average separation of 100 nm if the Oligo-dT wereapplied at 2 uM prior to 100-fold air dying, or conversely, if theOligo-dT were applied at 50 uM, in the original water-containing phasethe pore size upon drying could be reduced to about 35 nm.

While still water-free, due to the dense proximity of Oligo-dT strands,photochemical crosslinking will be efficient: between Oligo-dT strandsand also between Oligo-dT and the protein (FIG. 1B). Interestingly, at250 ug/ml, the same calculation suggest that protein molecules will onaverage “pile” to form a layer on the microarray surface that is about12 protein molecules deep within the water depleted, 1 um thickmicroarray spot. Thus, FIG. 1B represents about 200 um (about ⅕^(th)) ofsuch a 1 um-thick desiccated microarray spot. Upon rehydration of that 1um thick Oligo-dT+protein layer, wetting will cause the layer to swell,the separation between protein molecules to increase, thus preparing theprotein, while still entrapped in the crosslinked Oligo-dT matrix, forsubsequent microarray-based binding interaction with water-solubleanalytes of interest.

Example 3 Protein Attachment to Solid Surfaces

The present invention is novel in that protein is not linked to thesurface directly, nor to a preformed polymer field, or to pores in thesolid support or by adsorption to the microarray surface. Instead, theprotein is applied to the microarray surface with Oligo-dT, whichalthough not a high polymer, forms an extended polymeric matrix within amicroarray spot, subsequent to controlled evaporative concentration,followed by photochemical crosslinking.

The physical and chemical entrapment of protein within that Oligo-dTmatrix is created locally, only within the spots comprising sites ofmicroarray fluid deposition, thereby allowing site-addressable,self-assembly of a 3 dimensional protein structure such as that in FIGS.1A-1B. The key components of the invention are the length of theOligo-dT (typically @50bases), the ratio of Oligo-dT to protein(typically 1/1 to 10/1 on a mole basis), and the ability to control thefinal concentration of protein and Oligo-dT after ordinary evaporation,by including water-miscible, non-volatile solvents and solutes,typically at 1% by mass, so that the protein & Oligo-dT will concentrate@100-fold prior to UV crosslinking.

A number of such water-soluble non-volatile solvents and solutes may beused, but they must all share the property that, upon evaporative waterloss, and, importantly, protein remains soluble and in a stable foldedform, i.e., does not denature, in the water-depleted state. There areseveral such solvents and solutes of that kind, including glycerol,propanediol, butanediol, trehalose, sorbitol, sucrose and mixturesthereof which are known to retain protein folding even when the proteinremains in them with little to no added water.

A Bovine Serum Albumin Microarray

The present invention describes using bovine serum albumin (BSA) as theprotein in a microarray. This BSA has been produced with a biotinmodification, so that streptavidin phycoerythrin (SAPE) can bind to thebiotin of it, and after imaging of the green PE fluorescence (532 nm)serve to localize the BSA protein on the microarray surface. The BSA isapplied to the surface as a solution with CY-5 labelled Oligo dT (50bases long) along with an excess of unlabeled Oligo-dT (50 bases long)which may be imaged via the red CY-5 fluorescence (635 nm). Table IIIprovides a detailed composition of that microarray.

TABLE III Bovine Serum Albumin (BSA) microarray design to yield data inFIGS. 2A-2B Left 6 × 13 segment of 12 × 12 Microarray [BSA] 250 100 5020 250 100 conc μg/ml μg/ml μg/ml μg/ml μg/ml μg/ml OligoT 50-mer 50-mer50-mer 50-mer 50-mer 50-mer 100 μM 100 μM 100 μM 100 μM 25 μM 25 μMglycerol glycerol glycerol glycerol glycerol glycerol OligoT 50-mer50-mer 50-mer 50-mer 50-mer 50-mer 25 μM 25 μM 25 μM 25 μM 100 μM 100 μMG-PD G-PD G-PD G-PD G-PD G-PD OligoT 50-mer 50-mer 50-mer 50-mer 50-mer50-mer 100 μM 100 μM 100 μM 100 μM 25 μM 25 μM trehalose trehalosetrehalose trehalose trehalose trehalose OligoT 50-mer 50-mer 50-mer50-mer control control 25 μM 25 μM 25 μM 25 μM G-S G-S G-S G-S OligoT50-mer 50-mer 50-mer 50-mer 50-mer 50-mer 100 μM 100 μM 100 μM 100 μM 25μM 25 μM glycerol glycerol glycerol glycerol glycerol glycerol OligoT50-mer 50-mer 50-mer 50-mer 50-mer 50-mer 25 μM 25 μM 25 μM 25 μM 100 μM100 μM G-PD G-PD G-PD G-PD G-PD G-PD OligoT 50-mer 50-mer 50-mer 50-mer50-mer 50-mer 100 μM 100 μM 100 μM 100 μM 25 μM 25 μM trehalosetrehalose trehalose trehalose trehalose trehalose OligoT 50-mer 50-mer50-mer 50-mer control control 25 μM 25 μM 25 μM 25 μM G-S G-S G-S G-SOligoT 50-mer 50-mer 50-mer 50-mer 50-mer 50-mer 100 μM 100 μM 100 μM100 μM 25 μM 25 μM glycerol glycerol glycerol glycerol glycerol glycerolOligoT 50-mer 50-mer 50-mer 50-mer 50-mer 50-mer 25 μM 25 μM 25 μM 25 μM100 μM 100 μM G-PD G-PD G-PD G-PD G-PD G-PD OligoT 50-mer 50-mer 50-mer50-mer 50-mer 50-mer 100 μM 100 μM 100 μM 100 μM 25 μM 25 μM trehalosetrehalose trehalose trehalose trehalose trehalose OligoT 50-mer 50-mer50-mer 50-mer control control 25 μM 25 μM 25 μM 25 μM G-S G-S G-S G-SRight 6 × 13 segment of 12 × 12 Microarray [BSA] 50 20 250 100 50 20conc μg/ml μg/ml μg/ml μg/ml μg/ml μg/ml OligoT 50-mer 50-mer 50-mer50-mer 50-mer 50-mer 25 μM 25 μM 100 μM 100 μM 100 μM 100 μM glycerolglycerol G-PD G-PD G-PD G-PD OligoT 50-mer 50-mer 50-mer 50-mer 50-mer50-mer 100 μM 100 μM 25 μM 25 μM 25 μM 25 μM PD PD PD PD PD PD OligoT50-mer 50-mer 50-mer 50-mer 50-mer 50-mer 25 μM 25 μM 100 μM 100 μM 100μM 100 μM trehalose trehalose G-S G-S G-S G-S OligoT OligoT 50-mer50-mer 50-mer 50-mer 50-mer 50-mer 25 μM 25 μM 100 μM 100 μM 100 μM 100μM glycerol glycerol G-PD G-PD G-PD G-PD OligoT 50-mer 50-mer 50-mer50-mer 50-mer 50-mer 100 μM 100 μM 25 μM 25 μM 25 μM 25 μM PD PD PD PDPD PD OligoT 50-mer 50-mer 50-mer 50-mer 50-mer 50-mer 25 μM 25 μM 100μM 100 μM 100 μM 100 μM trehalose trehalose G-S G-S G-S G-S OligoTOligoT 50-mer 50-mer 50-mer 50-mer 50-mer 50-mer 25 μM 25 μM 100 μM 100μM 100 μM 100 μM glycerol glycerol G-PD G-PD G-PD G-PD OligoT 50-mer50-mer 50-mer 50-mer 50-mer 50-mer 100 μM 100 μM 25 μM 25 μM 25 μM 25 μMPD PD PD PD PD PD OligoT 50-mer 50-mer 50-mer 50-mer 50-mer 50-mer 25 μM25 μM 100 μM 100 μM 100 μM 100 μM trehalose trehalose G-S G-S G-S G-SOligoT

To the combination of BSA and Oligo-dT was added any one of a number ofnon-volatile, non-aqueous solvents and solutes including glycerol (G),glycerol-propanediol (GP) trehalose (T) and glycerol-sorbitol (GS). Theresulting solution was printed onto either amino-silane coated orepoxysilane coated glass microarray substrates, using contact printingto form 100 um diameter spots, roughly 200 um on center. The printvolume (@500 pL) was found to air-dry within 10 min, as assessed byfluorescent imaging of the microarray surface (Axon Imager).

Subsequent to air drying, the microarrays were crosslinked by exposureto 300 mJ of UV light (Stratolinker) to induce thymidine photochemicalcrosslinking via the thymidine moieties of the Oligo-dT. Subsequent tocrosslinking, the slides were subjected to two 5 minute washing stepswith a Tween 20 containing buffer, followed by administration of SAPE,to bind to the biotinylated BSA, so that it may be localized andquantitated by fluorescent imaging. In some cases, the SAPE staining wasperformed with 1% casein in the SAPE binding buffer, to provide foradditional blocking of spurious SAPE binding to regions not modifiedwith BSA. Subsequent to completion of the binding step (performed atroom temp) a series of washes in protein free binding buffer and twowater washes were performed (also at Room Temp) followed by air dryingand imaging on an Axon laser scanner: at 532 nm (green for SAPE) and 635nm (red, for Cy-5 labelled Oligo-dT).

A microarray design was used which presented various combinations ofBSA, Oligo-dT and non-aqueous solvent and solute concentration (TableIII). Representative images obtained of those microarrays, after bindingand washing are presented in FIG. 2A (formed on an amino silane surface)or in FIG. 2B (formed on an epoxysilane surface).

As can be seen from the (red) CY-5 labelled Oligo-dT images themicroarray printing and UV crosslinking procedure has permanently linkedthe Oligo-dT complement to the microarray surface on both an underlyingaminosilane (FIG. 2A) and epoxysilane surface (FIG. 2B). As seen fromthe corresponding images generated via SAPE staining of biotinylated BSA(FIGS. 2A-2B, Right panels) the microarray printing and UV crosslinkingprocedure has also permanently linked the BSA protein to the surface, sothat it may bind to SAPE.

Those image data have been quantified spot by spot and presented in FIG.3 and FIG. 4 as the ratio of SAPE/Cy5 signal intensity, which is ameasure of the ratio of BSA to Oligo-dT in each spot, as a function ofvariation in the several parameters described in Table III. FIG. 3describes those ratios when the microarray is printed upon theaminosilane coated microarray substrate and FIG. 4 the correspondingdata on the epoxysilane coated microarray substrate. Table IV providesthe test conditions under which the ratios in FIGS. 3 and 4 wereobtained. In Tables Va, Vb and Vc the numerical values used to generateFIGS. 3 and 4 are provided.

TABLE IV Sodium Biotinylated 50 mer Cy5 Abbre- Phosphate BSA Oligo-TOligo-T Adjuvant viation pH 8.4 (mM) (μg/ml) (μM) (μM) Glycerol G1 2 250100 1 1% G2 2 100 100 1 G3 2 50 100 1 G4 2 20 100 1 G5 2 250 25 1 G6 2100 25 1 G7 2 50 25 1 G8 2 20 25 1 Glycerol- GP1 2 250 100 1 PropanedioGP2 2 100 100 1 0.5%-0.5% GP3 2 50 100 1 GP4 2 20 100 1 GP5 2 250 25 1GP6 2 100 25 1 GP7 2 50 25 1 GP8 2 20 25 1 Propanediol PD1 2 250 100 11% PD2 2 100 100 1 PD3 2 50 100 1 PD4 2 20 100 1 PD5 2 250 25 1 PD6 2100 25 1 PD7 2 50 25 1 PD8 2 20 25 1 Trehalose T1 2 250 100 1 1% T2 2100 100 1 T3 2 50 100 1 T4 2 20 100 1 T5 2 250 25 1 T6 2 100 25 1 T7 250 25 1 T8 2 20 25 1 Glycerol- GS1 2 250 100 1 Sorbitol GS2 2 100 100 10.5%-0.5% GS3 2 50 100 1 GS4 2 20 100 1 GS5 2 250 25 1 GS6 2 100 25 1GS7 2 50 25 1 GS8 2 20 25 1 Control C1 150 250 0 1 Trehalose C2 150 250100 1 0.5%

TABLE Va SAPE @ 0.1 μg/ml 635 nm 635 nm SAPE/ Signal Signal 532 nm Cy5Test Intensity Intensity Signal SAPE Signal Condi- Post Post SAPEDecrease Signal Intensity tion Printing binding (%) Intensity Ratio G116955 9416 44 1206 0.13 G2 6330 4121 35 1206 0.29 G3 1037 753 27 10921.45 G4 1939 1665 14 970 0.58 G5 1832 991 46 1002 1.01 G6 534 337 371066 3.16 G7 504 366 27 1104 3.02 G8 254 138 46 199 1.44 GP1 22508 1328441 1110 0.08 GP2 11357 7742 32 1067 0.14 GP3 6701 4608 31 1109 0.24 GP4572 567 1 208 0.37 GP5 16316 7741 53 975 0.13 GP6 5105 2814 45 1072 0.38GP7 5534 3356 39 1088 0.32 GP8 2547 2142 16 935 0.44 PD1 65535 62387 5903 0.01 PD2 37757 13239 65 949 0.07 PD3 25895 8991 65 671 0.07 PD4 75424459 41 377 0.08 PD5 52806 26927 49 796 0.03 PD6 43311 15303 65 810 0.05PD7 33466 17870 47 972 0.05 PD8 35202 14819 58 952 0.06 T1 65535 9356 86805 0.09 T2 65535 16762 74 974 0.06 T3 65535 20414 69 423 0.02 T4 6553530171 54 353 0.01 T5 65535 10151 85 930 0.09 T6 65535 20582 69 991 0.05T7 65535 32239 51 991 0.03 T8 65535 36545 44 631 0.02 GS1 65535 38155 42761 0.02 GS2 11220 5398 52 696 0.13 GS3 2844 1649 42 711 0.43 GS4 56063358 40 779 0.23 GS5 32347 2626 92 907 0.35 GS6 7226 2089 71 605 0.29GS7 5039 1391 72 344 0.25 GS8 3797 1475 61 451 0.31 C1 65535 65535 0 7300.01 C2 65535 65535 0 609 0.01

TABLE Vb SAPE @ 0.1 μg/ml + 1% casein 635 nm 635 nm SAPE/ Signal Signal532 nm Cy5 Test Intensity Intensity Signal SAPE Signal Condi- Post PostSAPE Decrease Signal Intensity tion Printing binding (%) Intensity RatioG1 12131 7829 35 924 0.12 G2 5625 3932 30 961 0.24 G3 1081 891 18 7490.84 G4 1150 1140 1 647 0.57 G5 1326 822 38 937 1.14 G6 501 372 26 10712.88 G7 611 480 21 1077 2.24 G8 150 139 7 135 0.97 GP1 15734 10458 341177 0.11 GP2 7716 5523 28 1398 0.25 GP3 6435 4714 27 1415 0.30 GP4 137124 9 123 0.99 GP5 12493 6449 48 867 0.13 GP6 3741 2450 35 909 0.37 GP73796 2701 29 859 0.32 GP8 3034 2173 28 791 0.36 PD1 65535 65535 0 8350.01 PD2 49436 40764 18 847 0.02 PD3 15169 8673 43 665 0.08 PD4 31592420 23 401 0.17 PD5 50343 30750 39 1201 0.04 PD6 35607 14057 61 12270.09 PD7 18201 9594 47 1072 0.11 PD8 21455 10282 52 940 0.09 T1 655359538 85 799 0.08 T2 65535 15743 76 559 0.04 T3 65535 17025 74 193 0.01T4 65535 19488 70 381 0.02 T5 65535 9878 85 657 0.07 T6 65535 17010 74573 0.03 T7 65535 24874 62 662 0.03 T8 65535 31337 52 376 0.01 GS1 6488025578 61 1071 0.04 GS2 10017 5088 49 1120 0.22 GS3 1718 824 52 1112 1.35GS4 2662 1400 47 1143 0.82 GS5 23631 2382 90 814 0.34 GS6 6148 2138 65412 0.19 GS7 2023 686 66 193 0.28 GS8 2577 1150 55 135 0.12 C1 6553565535 0 609 0.01 C2 65535 65535 0 466 0.01

TABLE Vc No SAPE Test 635 nm Signal 635 nm Signal Signal Condi-Intensity Post Intensity Post Decrease tion Printing SAPE binding (%) G112204 7757 36 G2 4491 3106 31 G3 1123 881 22 G4 1107 909 17 G5 1164 74736 G6 471 349 26 G7 526 388 26 G8 149 115 23 GP1 17385 11358 35 GP2 83256067 27 GP3 6290 4843 23 GP4 158 143 9 GP5 8795 5222 41 GP6 3563 2312 35GP7 2999 2373 21 GP8 3017 2260 25 PD1 65535 65535 0 PD2 65328 65202 0PD3 17243 13924 19 PD4 1887 1389 26 PD5 42002 31619 25 PD6 29609 1624045 PD7 13903 6796 51 PD8 22972 9982 57 T1 65535 8988 86 T2 65535 8687 87T3 65535 9271 86 T4 58049 10853 81 T5 65535 7114 89 T6 65535 10515 84 T765535 12761 81 T8 65535 18387 72 GS1 65535 25670 61 GS2 10199 5146 50GS3 1652 789 52 GS4 2339 1011 57 GS5 21301 1977 91 GS6 6112 1904 69 GS72309 713 69 GS8 1217 397 67 C1 65535 65535 0 C2 65535 65535 0

The trends obtained from FIGS. 3 and 4 are instructive. Overall,Oligo-dT crosslinking to permanently affix it onto the surface isefficient for both surfaces, more-less independent of the supportingnon-aqueous solvent and solutes added. However, when the resultingSAPE/Cy-5 ratio was obtained, it was seen that glycerol (G)glycerol-propanediol (GP) and glycerol-sorbitol (GS) each provide forsubstantial biotin-BSA interaction with its cognatestreptavidin-phycoerythrin (SAPE) conjugate. In contrast, it is seenthat Trehalose (T) and to a lesser extent Propanediol (PD) provide forvery poor BSA interaction due to poor protein association to the surfaceor protein disruption on the surface or both.

The above Examples demonstrate that Oligo-dT mediated UV crosslinkingallows a protein such as BSA to be linked to a microarray surface toform, upon air-drying, a principally water free phase containing anumber of nonvolatile water miscible solvents and solutes. The data showthat, for BSA, certain solvent-solute pairs, e.g., glycerol and glycerolpropane diol, appear to be superior to trehalose and propandiol in thepresent case of BSA attachment to the microarray surface.

What is claimed is:
 1. A formulation to link protein to a solid support,comprising: one or more proteins; Oligo-dT; and one or morenon-volatile, water-soluble protein solvents, solutes or combinationthereof in an aqueous solution.
 2. The formulation of claim 1, whereinthe water soluble protein solvent comprises glycerol or the watersoluble protein solvent and solutes comprise glycerol and at least oneof sucrose, trehalose or sorbitol.
 3. The formulation of claim 2,wherein said sucrose, trehalose or sorbitol is present at a mass ratioof about 0.5:1 up to about 4:1 relative to glycerol.
 4. The formulationof claim 1, wherein the water soluble protein solvent comprisespropanediol or the water soluble protein solvent and solids comprisepropanediol and at least one of sucrose, trehalose or sorbitol.
 5. Theformulation of claim 4, wherein said sucrose, trehalose or sorbitol ispresent at a mass ratio of about 0.5:1 up to about 4:1 relative topropanediol.
 6. The formulation of claim 1, wherein the water solubleprotein solvents and solids comprise glycerol and propanediol and atleast one of sucrose, trehalose or sorbitol.
 7. The formulation of claim1, where the Oligo-dT is about 30 bases to about 100 bases in length andis contained in said formulation in a concentration of about 1 μg/ml toabout 100 μg/ml.
 8. The formulation of claim 1, where the protein isabout 50 kD to 250 kD in mass and is contained in said formulation in aconcentration of at least 10 μg/ml.
 9. The formulation of claim 1,wherein the solid support is an amino-silane layer disposed upon anunderlying surface.
 10. The formulation of claim 9, wherein saidunderlying surface is a metal surface, a glass surface or a ceramicsurface.
 11. The formulation of claim 1, wherein the solid support is anepoxy-silane layer disposed upon an underlying surface.
 12. Theformulation of claim 11, wherein said underlying surface is a metal. 13.The formulation of claim 1, wherein the non-volatile solvent and solutesare formulated in water at about 1% by mass.
 14. The formulation ofclaim 1, wherein the protein is an immunoglobulin, a glycoprotein, aviral protein, an intact virus, albumin, an HLA, or an enzyme.
 15. Aformulation to link protein to a solid support, comprising: one or moreproteins; Oligo-dT; and glycerol or glycerol and at least one ofsucrose, trehalose or sorbitol in an aqueous solution.
 16. Theformulation of claim 15, where the Oligo-dT is about 50 bases long andis contained in the formulation in a concentration of about 1 μg/ml toabout 100 μg/ml.
 17. The formulation of claim 15, where the protein isabout 50 kD to 250 kD in mass and is contained in the formulation in aconcentration of at least 10 μg/ml.
 18. The formulation of claim 15,wherein the solid support is an amino-silane layer or epoxy-silane layerdisposed upon an underlying surface comprising a metal surface, a glasssurface or a ceramic surface.
 19. The formulation of claim 15, whereinthe protein is an immunoglobulin, a glycoprotein, a viral protein, anintact virus, albumin, an HLA, or an enzyme.